Ta cloning vector pmd18 t

One MYB unigene and one bHLH unigene were selected from the mixed floral and foliar transcriptome database of anthurium^{29 (link)}, which were annotated as AtTT2-like and AtTT8-like transcription factors, respectively. The unigenes were designated as AaMYB3 (GenBank accession no. MH349476) and AabHLH1 (accession no. MH349477). The full-length cDNA of AaMYB3 and AabHLH1 was isolated from the “Tropical” spathe with reverse transcription (RT)-PCR using primers designed according to the transcriptome data: forward 5′-ATGGGCAGGAGACCCTGTT-3′, reverse 5′-CGCCATTACTTCACCCATTC-3′ for AaMYB3, and forward 5′-AGGAGGGGTAGTTGAGCAGGT-3′, reverse 5′-TCATGCTCTAAGCATGTCACGA-3′ for AabHLH1. PCR-amplified products were cloned into the T/A cloning vector pMD18-T (TaKaRa, Dalian, China) and sequenced. The full length of the amino acid sequences deduced for AaMYB3 and AabHLH1 were constructed using the ClustalX2 and the MEGA 5.05 programs (the Neighbor-Joining method with 1000 bootstrap replications) for sequence alignment and phylogenetic analysis, respectively.

Gel Extraction Mini Kit (Omega, USA) was used for DNA purification and recovery of the PCR products. Purified PCR products ligated with a TA cloning vector pMD18-T (TaKaRa, Japan) were transformed into competent E. coli cells strain JM109 (Beijing TransGen Biotech, PRC). Confirmation of clones containing recombinant plasmid was achieved by PCR and restriction enzyme digestion. The PCR conditions were the same as that for the above-mentioned PCR amplification. Three positive clones were randomly selected and cultured. Recombinant plasmids were sequenced at Shanghai Sang-gong Biological Engineering Technology & Services Co., Ltd (Shanghai, China).

E. coli DH5α, S. aureus, Rhizoctonia. solani Khün (rice)-10 and Sclerotinia. sclerotiorum (Lib.) de Bary-14., were obtained from the state key laboratory of agricultural microbiology of Huazhong Agricultural University, Wuhan-China. E. coli BL21 (DE3) and expression plasmid pET32a were purchased from Novagen (Piscataway, NJ, USA). TA cloning vector pMD18-T, T4 DNA ligase, and Taq DNA polymerase were purchased from TaKaRa Biotechnology (TaKaRa, Japan). AxyPrep DNA Gel Extraction Kit is the product of Axygen Scientific Inc (USA). TRIzol reagent and TransScript^™III first-Strand cDNA Synthesis SuperMix were obtained from Invitrogen (USA).